Project Summary/Abstract Ca[2+] is the principal second messenger in translating electrical signals (i.e. action potentials) into mechanical process in the heart (i.e. contractions). This highly orchestrated process of excitation-contraction coupling (ECC) is achieved through a number of transporters, pumps, and ion channels on the sarcolemmal membrane as well as in the sarcoplasmic reticulum (SR), resulting in the Ca[2+] waves observed during a beat-to-beat cycle. Modulation of these Ca[2+] handling proteins by the ubiquitously expressed Ca[2+]/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) have been found to play an increasingly important role in the heart. Cardiomyocytes signaling during ECC is described by the flux in intracellular [Ca[2+]]i concentration. During the increase in [Ca[2+]]i Ca[2+] bound calmodulin (Ca-CaM) binds to and activates CaMKII and CaN; thereby phosphorylating or dephosphorylating several important Ca[2+] handling proteins with multiple functional consequences within the cell. CaMKII and CaN signaling are independent of each other, but can also be antagonistic in the phosphorylation signaling cascade. CaMKII activity and expression were shown to be increased in human cardiac hypertrophy, heart failure, and animal models of heart failure. Inhibiting CaMKII activity in animal models of heart failure reduces or prevents pathology. The inhibition of CaN has demonstrated beneficial effect in animal models of heart failure; however the signaling mechanisms are not understood and need further investigation. Thus, these Ca-CaM-dependent pathways are critical nodal points and potentially valuable therapeutic targets in the genesis and treatment of heart failure and arrhythmias. The specific aims are focus on localization and translocation (A) and activation dynamics (B) of the Ca-CaMCaMKII- CaN signaling pathways with the following hypothesizes: (1A) Ca-CaM localization and translocation are induced by different stimuli. (1B) Ca-CaM signaling dynamics differ in key myocyte locations. (2A) CaMKIIdelta{B} vs. delta{C} differ in localization and translocation. (2B) CaMKII activation in myocytes differs locally and integrates to cause memory. (3A) CaN localizes at the Z-line, but translocates with local Ca[2+] signals. (3B) Kinetics and location of CaN activation differ from CaMKII in adult cardiomyocytes. The Bers Lab has generated a set of fluorescently tagged proteins to monitor localization and translocation of CaM, CaMKII and CaN. There are also FRET based activity reporters for Ca-CaM, CaMKII and CaN. These constructs will be transfected into isolated adult cardiomyocytes for live cell imaging and the use of FRAP and TIRF will be employed to investigate the dynamics of the signaling pathways. After characterization of the Ca-CaM-CaMKIICaN pathway in heathly heart cells, cardiomyocytes from heart failure models will be used to investigate pathological changes. This will greatly enhance our understanding of how CaMKII and CaN activity is regulated in the heart by both pathological and physiological signaling pathways. This will help to identify novel signaling cascades and thereby identify new therapeutic targets for the treatment of heart failure.